Figure 3: Affinity-directed HP1α dimerization allows investigating multivalent chromatin binding.
From: Multivalency governs HP1α association dynamics with the silent chromatin state
(a) Strategy to synthesize HP1αcdm employing the PxVxI peptide P2 in an affinity-directed dual-EPL reaction (PDB: 3Q6S, ref. 39). (b) One-pot production of Atto532-labelled HP1αcdm: HP1α(1–177), fused to the NpuN-split intein was bound to NpuC-beads and eluted with ligation buffer containing the hSgoL1 peptide (carrying two cysteinyl-lysines and an Atto532 dye), yielding labelled HP1αcdm (Atto532). Pure HP1αcmd was obtained after gel-filtration purification. Gel annotation: I=input; FT=flowthrough of column; W1-2=column washes; E1-3=elution fractions; Fn=final protein after gel filtration. (c) Time trace of fluorescence intensity (blue) from a single chromatin array fitted by a step function (red) and showing transient HP1αcdm binding events (at 0.5 nM). (d) HP1αcdm dissociation kinetics (100 chromatin arrays, 105 frames each) fitted by a double-exponential function (fit: τoff,1=0.33±0.01 s, τoff,2=3.40±0.53 s). (e) HP1αcdm association kinetics over 30 chromatin arrays, fitted by a single-exponential function (fit: λon=7.45±1.87 s). (d,e) For all fit results, see Table 1.
